Summary Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. Chromosomal organization involves two principal mechanisms: topological maintenance and protein-mediated packaging of the DNA. The former prevents entanglement by regulating the topology of the DNA, resolving unwanted catenanes and knots. The latter shapes the conformation of chromosomes, increasing the efficiency of any particular macromolecular transaction. In this proposal, we investigate these fundamental processes of chromosome organization by testing two hypotheses that center on the role of the interaction of topoisomerase IV (Topo IV), the enzyme that unlinks the daughter chromosomes, with the bacterial condensin, MukB; the mechanism by which the condensin itself acts on DNA; and the role of topoisomerase III (Topo III) in chromosome segregation in Escherichia coli. We showed that the interaction between the ParC subunit of Topo IV and MukB results in stimulation of only intramolecular reactions (such as superhelical DNA relaxation and DNA knotting) and not intermolecular reactions (such as DNA decatenation) catalyzed by Topo IV, suggesting that the interaction does not play a role in separation of the linked chromosomes directly. We will test our hypothesis that MukB and Topo IV act to condense the chromosome by bringing distal segments of the DNA together by examining the effects of ablating this interaction on chromosome dynamics in vivo, characterizing biochemically the effects of ParC on formation of the complete MukBEF condensin and on MukB-modulation of DNA topology, and characterizing a new MukB-mediated reaction that we have discovered: catenation of gapped DNA rings in the presence of Topo III. This latter reaction is more likely to accurately reflect the action of MukB to condense DNA in vivo than any of the other MukB-mediated, DNA topology-altering reactions that have been described. We showed that deletion of Topo III sensitizes cells to the type II topoisomerase inhibitor novobiocin, even when DNA gyrase is resistant to the drug, and that topB mutations combined with temperature-sensitive mutations in the Topo IV genes were synthetically lethal and showed chromosome segregation defects at semi-permissive temperatures. We have also shown that Topo III co-localizes with replisome components in vivo. We propose that Topo III participates in chromosome segregation by unlinking precatenanes (windings of the two partially replicated sister duplexes about each other) as they form at the replication fork. We will test this hypothesis by asking whether Topo III tracks with the replication fork, determining the manner by which it associates with the replisome, and the consequences on sister chromosome cohesion (which is thought to be mediated by precatenation) of deleting Topo III from the cell.